IL-33/ST2L signaling alleviates diabetic nephropathy by regulating endoplasmic reticulum stress and apoptosis.

OBJECTIVE: Diabetic nephropathy (DN) is a serious chronic complication of diabetes mellitus (DM). Endoplasmic reticulum (ER) stress is an important factor in the regulation of pathological processes in DN, and excessive ER stress can lead to apoptosis. Although the IL-33/ST2 axis is known to be involved in diabetic kidney disease or related nephropathy, its role and molecular mechanisms remain poorly understood in terms of DN. The purpose of this study was to investigate the effects of IL-33/ST2 signaling on DN and to characterize the roles that ER stress and apoptosis play in DN. METHODS: To investigate this study, mice were randomly assigned into DN (induced by 0.1% STZ) and Control groups. Biochemical indices (FBG, BUN, UPR, UCE) were measured in serum and urine samples to reflect blood glucose and kidney damage. Quantitative real-time PCR, western blot, and immunofluorescence were used to assess gene and protein expression of the IL-33/ST2 axis and ER stress relative signaling molecule. Apoptosis was analyzed by flow cytometry. RESULTS: IL-33 levels are significantly increased in the kidneys of patients and mice with DN. Double immunofluorescence staining showed that IL-33 colocalized with CD31-positive endothelial cells. Treatment with IL-33 attenuated kidney injury in Streptozotocin (STZ)-treated mice. In vitro, we showed that IL-33 attenuated ER stress and apoptosis in glomerular endothelial cells. However, sST2 treatment significantly reversed these effects of IL-33. CONCLUSION: Together, these data suggest that IL-33/ST2 signaling mitigates STZ-induced renal damage, partly at least, by suppressing ER stress and apoptosis. Therefore, IL-33 may be an effective therapeutic target in DN.

Effect and mechanism of TFEB on pyroptosis in HK-2 cells induced by high glucose.

As a newly discovered way of cell death, pyroptosis has been gradually discovered in acute and chronic kidney disease. Existing studies have shown that reactive oxygen species (ROS) can induce pyroptosis and release a large number of inflammatory mediators, resulting in kidney damage. As a transcription factor, transcription factor EB(TFEB) can regulate mitochondrial energy metabolism, reduce the production of ROS, and reduce the inflammatory damage of vascular endothelial cells. In a high-glucose environment, whether TFEB can regulate oxidative stress in HK-2 cells, thereby reducing pyroptosis, has not yet been studied. This study found that in HK-2 cells, with the prolongation of high concentration glucose stimulation, the expression level of TFEB showed a trend of first increasing and then decreasing; and nuclear translocation of TFEB expression occurred within 24 h. In high-glucose environment, the expression of pyroptosis-related proteins gradually increased over time, while the expression of anti-oxidative stress proteins superoxide dismutase2(SOD2)and NAD(P)H: quinone oxidoreductase 1(NQO1) showed a trend of first increasing and then decreasing. After TFEB was transfected with overexpression plasmid, the expression levels of SOD2 and NQO1 increased significantly, and the expression of pyroptosis-related proteins decreased. Observed under a confocal microscope after Mitosox red staining, the expression of ROS in the TFEB overexpression group decreased. After down-regulating the expression of TFEB, the expression of ROS increased. The research results suggested that in HK-2 cells in the high glucose environment, TFEB may affect the pyroptosis by regulating the expression of antioxidant enzymes SOD2 and NQO1, which provides a new therapeutic idea for the treatment of diabetic nephropathy.

Usage of Negative Pressure Wound Therapy in Pseudoepitheliomatous Hyperplasia Secondary to Burn Injury: A Case Series.

Pseudoepitheliomatous hyperplasia (PEH) is a reactive epithelial proliferation secondary to a wide range of stimuli, including traumatic injury, inflammation, infection, and tumors of the skin. PEH secondary to burn injury is rarely reported. We report three cases of PEH patients after burn injury. All three cases were confirmed with the existence of bacterial infection, and all these cases were second- or third-degree burns. All three patients were treated with negative pressure wound therapy after wound debridement or tangential excision. All the wounds healed without split-thickness skin grafting and recurrence.

Usage of intermingled skin allografts and autografts in a senior patient with major burn injury.

Mortality rate in older adults following extensive burn injury is extremely high, and management of these patients is challenging. One of the main problems is that autologous split-thickness skin grafts are scarce and the wounds cannot be covered quickly and effectively. Intermingled skin grafting is a low-tech and economic method, which not only maximizes the use of precious autologous skin but also prevents the wounds from infection and consumption. Herein we present a case of extensive burn injury in a 68-year-old female successfully treated with intermingled skin grafting. The patient was accidentally burned by gas flame, resulting in a major burn injury covering 80% of her total body surface area. Early burn wound excision was performed and the wound was temporarily covered with irradiated porcine skin in the first week after injury. Autologous stamp-like skin grafts were applied to the wound bed 4 weeks after injury. In this operation, the results were not satisfactory. The take rate of the skin grafts is only about 50%. We covered the wounds with intermingled skin allografts and autografts 8 weeks after injury: autografts (0.5 cm × 0.5 cm) + fresh close relative's allografts (1 cm × 1 cm) + cryopreserved allografts (2 cm × 2 cm).

Anti-hyperlipidemic, Anti-inflammatory, and Ameliorative Effects of DRP1 Inhibition in Rats with Experimentally Induced Myocardial Infarction.

This study aims to investigate the biological role of DRP1 in myocardial infarction (MI) in concert with hyperlipidemia (HL). Based on the available literatures, 10 genes related to MI with HL (HL-MI) were screened and detected in clinical samples. High-fat diet (HFD) was used to establish HL rat models, after which the rats were subcutaneously injected with PBS or isoproterenol hydrochloride to induce acute MI. Then, rats with HL-MI were injected with pcDNA3.1, pcDNA3.1-DRP1, sh-NC, or sh-DRP1. Serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) were measured. Cardiac function was evaluated by detecting left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF). Infarct size and histopathological changes were assessed as well as myocardial apoptosis and collagen deposition. The concentration of IL-6, IL-1ß, and TNF-α in rat serum and cardiac tissues was also measured by ELISA. Mitochondrial function was shown by measuring the morphology, mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) level. Pro-apoptotic proteins (Bax, caspase-1, and cleaved caspase-1) and NLRP3 inflammasome activation were also assessed. The expressions of the 10 genes were measured in clinical samples and DRP1 was selected for further experiments with significantly upregulated expression in MI patients. HFD-induced rats showed increased body weight, concurrent with higher levels of TG, TC, and LDL-C and lower HDL-C level. Compared with the BD-PBS group, the HFD-PBS group presented higher mRNA and protein expression levels of DRP1, exacerbated cardiac functions, enlarged infarct size, loss of cardiomyocytes, and disordered island cardiomyocytes. In the HL-MI rat model, injection of pcDNA3.1-DRP1 enhanced the levels of serum lipids and inflammation cytokines, induced loss of a number of cardiomyocytes and collagen deposition, and decreased LVFS and LVEF, while injection of sh-DRP1 ameliorated myocardial injuries, inflammation, and cardiomyocyte apoptosis and fibrosis. In coronary artery endothelial cells from the rats, loss of MMP was observed in the HFD-MI, LV-NC, LV-DRP1, and sh-NC groups and concomitantly, the sh-DRP1group showed increased MMP and decreased levels of mitochondrial ROS, cytochrome C, pro-apoptotic proteins, and NLRP3. Inhibition of DRP1 markedly suppressed HL, systematic inflammation, and myocardial injuries induced by HL-MI through partly restoring mitochondrial function and reducing NLRP3 expression.

[Analysis on the diagnosis and treatment of necrotizing fasciitis complicated with sepsis].

OBJECTIVE: To recognize the characteristics of necrotizing fasciitis patients complicated with sepsis and summarize the experience the treatment. METHODS: A retrospective study was conducted. The clinical data of 57 patients with necrotizing fasciitis complicated with sepsis admitted to Guangdong Provincial People's Hospital from July 2009 to December 2019 was analyzed by collecting such factors as gender, age, complications, infection sites, pathogens, surgery information, treatment options and outcome. The patients were divided into debridement group (n = 14) and control group (n = 43) according to whether the debridement was completed within 48 hours of admission, and the mortality during hospitalization between the two groups was compared. A telephone follow-up had been done to record the long-term outcome of these patients. RESULTS: Among 57 patients with necrotizing fasciitis complicated with sepsis, there were 43 males and 14 females with the average age of (57.9±12.1) years old. Most of the underlying diseases were diabetes mellitus (70.17%), other diseases included hypertension (8.77%), tumor chemotherapy (7.02%), liver disease (hepatitis, cirrhosis, 7.02%), coronary artery heart disease (3.51%), systemic lupus erythematosus (3.51%), etc. Most of the infection site was lower limbs (71.93%). There were 78 pathogens cultured in 57 patients, in which 52 were non-drug resistant bacteria (66.67%), and 26 were drug resistant bacteria (33.33%). There were 40 Gram positive (G+) bacteria (51.28%), 29 Gram negative (G-) bacteria (37.18%), 8 fungi (10.26%) and 1 mixed bacteria (1.28%). Finally, of 57 patients, 46 patients were cured, and 11 patients died with hospital mortality of 19.30%. Among 57 patients, the hospital mortality in the debridement group was significantly lower than that in the control group [0% (0/14) vs. 25.58% (11/43), P < 0.05]. Among the 46 cured patients, 11 had accepted amputations, accounting for 23.91%. In December 2020, 43 patients who were cured (3 patients were lost to follow-up) were followed up by telephone. Twenty-three patients were completely self-care, 9 patients were partly self-care, 8 patients were completely unable to take care of themselves, and 3 patients died. CONCLUSIONS: Necrotizing fasciitis with sepsis mostly occurs in people with weakened immunity, and has a high mortality and disability rate. Early identification and active surgical debridement may be the key to improve the treatment effect.

Electroacupuncture alleviates intestinal inflammation and barrier dysfunction by activating dopamine in a rat model of intestinal ischaemia.

BACKGROUND: To investigate whether the mechanism underlying the anti-inflammatory effects of electroacupuncture (EA) at ST36 involves dopamine (DA) and its receptor and whether it is mediated by the vagus nerve in a rat model of intestinal ischaemia-reperfusion (I/R) injury. METHODS: Rats were subjected to gut ischaemia for 30 min and then received EA for 30 min with or without abdominal vagotomy or intraperitoneal administration of butaclamol (D1 receptor antagonist) or spiperone (D2 receptor antagonist). Plasma levels of DA and tumour necrosis factor (TNF)-α were assessed 1 or 4 h after reperfusion. Myeloperoxidase (MPO) activity and malondialdehyde (MDA) content in intestinal tissues were assessed using enzyme-linked immunosorbent assay (ELISA) kits. Intestinal tissue injury was assessed by observation of the pathological lesions and permeability to 4 kDa fluorescein isothiocyanate (FITC)-dextran. RESULTS: EA significantly increased levels of DA and lowered levels of TNF-α. EA also inhibited intestinal levels of MPO and MDA and intestinal tissue injury and decreased intestinal permeability to FITC-dextran. Abdominal vagotomy and intraperitoneal administration of butaclamol (but not spiperone) inhibited the effects of EA. CONCLUSION: These findings suggest that EA at ST36 could attenuate intestinal I/R-induced inflammatory injury and that the underlying mechanism may involve EA-induced increases in levels of DA, mediated by the vagus nerve and D1 receptors.

Calcitonin gene-related peptide inhibits angiotensin II-induced NADPH oxidase-dependent ROS via the Src/STAT3 signalling pathway.

We had previously demonstrated that the calcitonin gene-related peptide (CGRP) suppresses the oxidative stress and vascular smooth muscle cell (VSMC) proliferation induced by vascular injury. A recent study also indicated that CGRP protects against the onset and development of angiotensin II (Ang II)-induced hypertension, vascular hypertrophy and oxidative stress. However, the mechanism behind the effects of CGRP on Ang II-induced oxidative stress is unclear. CGRP significantly suppressed the level of reactive oxygen species (ROS) generated by NADPH oxidase in Ang II-induced VSMCs. The Ang II-stimulated activation of both Src and the downstream transcription factor, STAT3, was abrogated by CGRP. However, the antioxidative effect of CGRP was lost following the expression of constitutively activated Src or STAT3. Pre-treatment with H-89 or CGRP8-37 also blocked the CGRP inhibitory effects against Ang II-induced oxidative stress. Additionally, both in vitro and in vivo analyses show that CGRP treatment inhibited Ang II-induced VSMC proliferation and hypertrophy, accompanied by a reduction in ROS generation. Collectively, these results demonstrate that CGRP exhibits its antioxidative effect by blocking the Src/STAT3 signalling pathway that is associated with Ang II-induced VSMC hypertrophy and hyperplasia.

Calcitonin gene-related peptide attenuates angiotensin II-induced ROS-dependent apoptosis in vascular smooth muscle cells by inhibiting the CaMKII/CREB signalling pathway.

Apoptosis is associated with various cardiovascular diseases. CGRP exerts a variety of effects within the cardiovascular system, and protects against the onset and development of angiotensin (Ang) II-induced vascular dysfunction and remodelling. However, it is not known whether CGRP has a direct effect on Ang II-induced apoptosis in vascular smooth muscle cells (VSMCs), and the mechanism underlying the anti-apoptotic role remains unclear. In this study, CGRP significantly suppressed reactive oxygen species (ROS) and apoptosis in Ang II-induced VSMCs. In VSMCs pre-treated with a CGRP receptor antagonist (CGRP8-37), the CGRP-mediated inhibition of Ang II-induced ROS and apoptosis was completely abolished. Moreover, pre-treatment with N-acetyl-L cysteine (NAC), an ROS scavenger, blocked the effects of CGRP on Ang II-induced apoptosis. In addition, the activation of CaMKII and the downstream transcription factor CREB stimulated by Ang II was abrogated by CGRP. Importantly, in both CGRP and NAC-treated VSMCs, CGRP failed to further attenuate CaMKII and CREB activation. The results demonstrate that CGRP attenuated Ang II-induced ROS-dependent apoptosis in VSMCs by inhibiting the CaMKII/CREB signalling pathway.

Valproic Acid Treatment Inhibits Vasopermeability and Improves Survival in Rats With Lethal Scald Injury.

The aim of this study was to examine whether administration of valproic acid (VPA), a histone deacetylase inhibitor, inhibits proinflammatory mediators and ameliorate visceral vasopermeability both in a rat model of major burn, and also in rat cultured endothelial cells stimulated with permeability evoking mediators. SD rats were subjected to a 50% TBSA full-thickness scald injury, and treated with either saline or VPA (300 mg/kg) intraperitoneally. Pulmonary vascular endothelial growth factor (VEGF), myeloperoxidase (MPO), pulmonary microvascular permeability, water content, and acetylation of histone H3K9 of lungs were evaluated. In addition, pulmonary microvascular endothelial cells (PMECs) from male SD rats were cultured. With then, MPO, VEGF, histone acetylation, and the permeability of PMECs were investigated. Lethal scald injury resulted in a significant increase in microvascular permeability and water content of lung, accompanied by a significant elevation of the content of VEGF and activity of MPO, and a decrease of histone acetylation. VPA treatment significantly alleviated the microvascular permeability and water content of lung, lowered the levels of VEGF and MPO, and promoted acetylation of histone H3K9 following scald injury. Moreover, VPA reduced permeability of monolayer PMECs subjected to scald serum challenge, reduced the level of MPO and VEGF in supernatants, and promoted acetylation of histone H3K9 in PMECs. These results indicated that VPA can protect pulmonary microvascular endothelial barrier, alleviate proinflammatory mediators-evoked vascular hyperpermeability and tissue edema and improve the survival rate of rats subjected to lethal scald injury.

[Protective effects of valproic acid on gut barrier function after major burn injury and its mechanism].

OBJECTIVE: To investigate the potential protective effects of valproic acid (VPA) on gut barrier function after major burn injury in rats and its mechanism. METHODS: Forty male Sprague-Dawley (SD) rats were divided into sham + normal saline (NS), sham + VPA, scald + NS, and scald + VPA groups, with 10 rats in each group. Rat with 55% total body surface area (TBSA) third-degree severe-burns model was reproduced by immersing into 80 °C water, and the rats in sham groups were given sham-burns by immersing into 37 °C water. The rats after severe-burns were immediately treated with 0.25 mL of 300 mg/kg VPA or NS by subcutaneous injection. Rats were sacrificed at 2 hours and 6 hours after injury, and abdominal aortic blood and ileal tissue were harvested. The levels of vascular endothelial growth factor (VEGF) were determined by enzyme-linked immunosorbent assay (ELISA). The intestinal permeability was evaluated by fluorescein isothiocyanate-dextran (FITC-dextran) determination. The histomorphological changes in gut barrier were evaluated by Chiu grading system. Levels of acetylated lysine at the ninth position of histone 3 protein (Ac-H3K9), hypoxia-inducible factor 1α (HIF-1α), zona occludens 1 (ZO-1) and myosin light chain kinase (MLCK) were determined by immunofluorescence staining and Western Blot. RESULTS: Compared with sham + NS group, rats in scald + NS group showed intestinal mucosal damage 2 hours after burn injury, as well as increased mucosal permeability, protein expression levels of HIF-1α, VEGF, MLCK, and lowered levels of AC-H3K9 and ZO-1. These changes were much more prominent at 6 hours after injury. VPA treatment significantly attenuated the burn-induced intestinal damage. Compared with scald + NS group, the protective effects in scald + VPA group was not evident at 2 hours after injury; however, intestinal damage was much less severe at 6 hours after injury (Chiu score: 2.03±0.27 vs. 3.12±0.15), intestinal permeability was significantly decreased [FITC-dextran (µg/L): 709±76 vs. 1 138±75], histone acetylation was enhanced [Ac-H3K9 (gray value): 1.55±0.12 vs. 0.48±0.12], ZO-1 degradation was significantly inhibited (gray value: 0.69±0.12 vs. 0.43±0.16), the protein expression levels of VEGF and MLCK were significantly down-regulated [VEGF (ng/mg): 51.7±3.7 vs. 71.2±4.3, MLCK (gray value): 1.98±0.20 vs. 2.80±0.24], while the HIF-1α protein expression levels were significantly reduced at both 2 hours and 6 hours after injury (gray value: 2.50±0.39 vs. 3.88±0.42 at 2 hours, 1.83±0.42 vs. 4.42±0.41 at 6 hours, all P < 0.05). CONCLUSIONS: Severe burn injury can induce histone deacetylation, ZO-1 degradation and intestinal barrier dysfunction. VPA can improve the levels of histone acetylation and ZO-1, and protect intestinal epithelial barrier function. These may probably be mediated through inhibiting HIF-1α and its downstream gene VEGF and MLCK.

[Protective effects of dimethyl sulfoxide on zymosan-insulted gut barrier impairment].

OBJECTIVE: To explore the effect of dimethyl sulfoxide(DMSO) on suppressing the release of gut inflammatory cytokine and re-store of the barrier impairment following zymosan-insulted systemic inflammatory response syndrome (SIRS). METHODS: S D rats were randomly divided into four groups:sham with administration of normal saline (SS group); sham with administration of DMSO (DS group); zymosan with administration of normal saline (ZS group); and zymosan with administration of DMSO (ZD group), each group includes two subgroups at 4 h and 24 h after surgery. At 4 h and 24 h after intraperitoneal injection of zymosan (750 mg/kg), the levels of intestinal inflammatory cytokines (tumor necrosis factor-αand interleukin-10) and the activity of diamine oxidase in plasma were examined. Intestinal injury was evaluated by using an intestinal histological score. RESULTS: DMSO suppressed the release of tumor necrosis factor-αand increased interleukin-10 levels in the intestine compared with the ZS group at the corresponding time points. DMSO decreased the level of diamine oxidase in plasma compared with the ZS group. DMSO restored the injury of intestinal villi and the gut injury score was significantly lower than that in the ZS group. CONCLUSIONS: DMSO can suppress the release of intestinal inflammatory cytokines and restore zymosan-insulted gut barrier impairment.

Pyruvate Is Superior to Citrate in Oral Rehydration Solution in the Protection of Intestine via Hypoxia-Inducible Factor-1 Activation in Rats With Burn Injury.

BACKGROUND: Recent studies have suggested that pyruvate-enriched oral rehydration solution (Pyr-ORS) may be superior to the standard bicarbonate-based ORS in the protection of intestine from ischemic injury. The aim of this study was to compare the effects of Pyr-ORS with citrate-enriched ORS (Cit-ORS) on the intestinal hypoxia-inducible factor-1 (HIF-1)-erythropoietin (EPO) signaling pathway for enteral rehydration in a rat model of burn injury. METHODS: Rats were randomly assigned to 4 groups (N = 20, 2 subgroups each: n = 10): scald sham (group SS), scald with no fluid resuscitation (group SN), scald and resuscitation with enteral Cit-ORS (group SC), and scald and resuscitation with enteral Pyr-ORS (group SP). At 2.5 and 4.5 hours after a 35% total body surface area (TBSA) scald, intestinal mucosal blood flow (IMBF), contents of HIF-1, EPO, endothelial nitric oxide synthase (eNOS), nitric oxide (NO), barrier protein (ZO-1), levels of serum diamine oxidase (DAO), and intestinal mucosal histology injury score were determined. RESULTS: Serum DAO activities in the scalded groups were significantly elevated, but less raised in group SP than in group SC, at 2.5 hours and at 4.5 hours after the scald. Further, group SP more profoundly preserved intestinal HIF-1 expression compared with group SC at the 2 time points. Compared with group SC, group SP had markedly elevated intestinal EPO, eNOS, and NO levels at the same time points, respectively (P < .05). Similarly, IMBF and ZO-1 levels were significantly higher in group SP than in group SC. Intestinal mucosal histopathological scores were statistically higher at 2.5 hours and 4.5 hours after scalding but were more attenuated in group SP than in group SC (P < .05). Immunofluorescence expression of intestinal mucosal ZO-1 was consistent with the above changes. The above parameters were also significantly different between groups SC and SN (all P < .05). CONCLUSION: Pyr-ORS provides a superior option to Cit-ORS for the preservation of intestinal blood flow and barrier function and the attenuation of histopathological alterations in enteral resuscitation of rats with burn injury. Its underlying mechanism may be closely related to the pyruvate in activation of intestinal HIF-1-EPO signaling cascades.

Association of the NAD(P)H oxidase p22 phox gene C242T polymorphism with type 2 diabetes mellitus, diabetic nephropathy, and carotid atherosclerosis with type 2 diabetes mellitus: A meta-analysis.

BACKGROUND: Several epidemiological studies have evaluated the association between the NAD(P)H oxidase p22 phox gene C242T polymorphism and the risk of type 2 diabetes mellitus (T2DM), diabetic nephropathy (DN), and carotid atherosclerosis with T2DM (CA), but the results are inconclusive. This meta-analysis was therefore designed to clarify these controversies. METHODS: Systematic searches were performed using electronic databases such as MEDLINE, PubMed, EMBASE, and China National Knowledge Infrastructure, as well as through manual searching of the references of identified articles. A total of 11 publications were eligible for this meta-analysis after running a search on the NAD(P)H oxidase p22 phox gene C242T polymorphism, including 7 with outcomes for T2DM, 7 with outcomes for DN, and 3 with outcomes for CA. The pooled odds ratio (OR) with a 95% confidence interval (CI) was calculated using a fixed effects model (FEM) or a random effects model (REM). Publication bias was tested by Begg's funnel plot analysis. Sensitivity analysis was also performed. RESULTS: The results showed a significant association between the NAD(P)H oxidase p22 phox gene C242T polymorphism and T2DM risk in the allelic model (REM: OR = 1.23, 95% CI = 1.06-1.43), additive model (FEM: OR = 1.61, 95% CI = 1.14-2.26), and recessive model (FEM: OR = 1.50, 95% CI = 1.10-2.05). A significant association was also observed for DN in the allelic model (REM: OR = 1.25, 95% CI = 1.06-1.47), additive model (FEM: OR = 1.61, 95% CI = 1.08-2.38), and dominant model (REM: OR = 1.26, 95% CI = 1.03-1.54). However, no association was observed for CA. Similar results were obtained in subgroup analysis based on ethnicity. CONCLUSIONS: Results of this meta-analysis suggest that the NAD(P)H oxidase p22 phox gene 242T allele might be associated with an increased risk of T2DM and DN, but not CA.

The extracellular matrix protein matrilin-2 induces post-burn inflammatory responses as an endogenous danger signal.

OBJECTIVE AND DESIGN: This prospective experimental study aims to investigate whether matrilin-2 is released from burn injury and induces post-burn inflammatory responses as an endogenous danger signal. SUBJECTS: Fifteen burn patients, 15 volunteers, 12 matrilin-2-deficient mice, 36 C57BL/6 mice and raw 264.7 cells. METHODS: Matrilin-2 levels were detected by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction. The inflammatory cytokines production in Matn2 deficient mice and wide type mice were detected by ELISA. Macrophages were activated by recombinant mouse MATN2 with or without adding anti-Toll-like receptor (TLR) 4 antibody. Student's t test and one-way analysis of variance were used for statistical analysis. RESULTS: The matrilin-2 levels in serum of burned patients were drastically elevated as compared to those of healthy controls. The matrilin-2 levels in burned mice were significantly increased than those of non-burned controls, whereas the matrilin-2 mRNA expression was not significantly changed after burn. In addition, Matn2 deficient mice showed remarkably less inflammatory cytokines production and less neutrophil infiltration in lung. Exogenous MATN2 induced potent expression of proinflammatory cytokines production in macrophages, which was inhibited by anti-TLR4 antibody. CONCLUSION: Matrilin-2 induces post-burn inflammatory responses as an endogenous danger signal, partly through a TLR4-mediated mechanism.

Apelin inhibits the activation of the nucleotide-binding domain and the leucine-rich, repeat-containing family, pyrin-containing 3 (NLRP3) inflammasome and ameliorates insulin resistance in severely burned rats.

BACKGROUND: Hyperglycemia with insulin resistance remains a challenging problem in severely burned patients. Recent studies indicated the involvement of the nucleotide-binding domain and the leucine-rich, repeat-containing family, pyrin-containing 3 (NLRP3) inflammasome in insulin resistance and a beneficial role of apelin in insulin resistance. Our aim was to investigate whether apelin inhibits the activation of the NLRP3 inflammasome and ameliorates insulin resistance in severely burned rats. METHODS: Male Wistar rats were subjected to a full-thickness burn injury comprising 40% of the total body surface area and were randomized to receive apelin, N(G)-methyl-L-arginine acetate salt (L-NMMA), and apelin plus treatments with L-NMMA. The following outcome measurements were assessed: apelin/APJ mRNA expression in white adipose tissue (WAT) and muscles, plasma apelin level, and activation of the NLRP3 inflammasome in WAT, Interleukin-1 ß, interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 levels in plasma, insulin resistance, survival rates, and endothelial nitric oxide synthase phosphorylation in soleus muscles. RESULTS: Severe burn induced a decreased expression of apelin/APJ mRNA in soleus muscles and a decrease in plasma apelin levels. Burn injury with apelin treatment restored plasma apelin level, inhibited NLRP3 inflammasome activity in WAT, and decreased inflammatory cytokine levels in plasma. Rats treated with apelin also showed improved insulin sensitivity and decreased mortality, accompanied by a remarkable induction of endothelial nitric oxide synthase phosphorylation in soleus muscle. Furthermore, the aforementioned effects of apelin were inhibited in part by treatment with L-NMMA. CONCLUSION: Apelin inhibits the activation of NLRP3 inflammasome, attenuates systemic inflammatory response, ameliorates insulin resistance, and promotes survival after severe burn, in part through an endothelial nitric oxide synthase-dependent pathway.

Electroacupuncture activates enteric glial cells and protects the gut barrier in hemorrhaged rats.

AIM: To investigate whether electroacupuncture ST36 activates enteric glial cells, and alleviates gut inflammation and barrier dysfunction following hemorrhagic shock. METHODS: Sprague-Dawley rats were subjected to approximately 45% total blood loss and randomly divided into seven groups: (1) sham: cannulation, but no hemorrhage; (2) subjected to hemorrhagic shock (HS); (3) electroacupuncture (EA) ST36 after hemorrhage; (4) vagotomy (VGX)/EA: VGX before hemorrhage, then EA ST36; (5) VGX: VGX before hemorrhage; (6) α-bungarotoxin (BGT)/EA: intraperitoneal injection of α-BGT before hemorrhage, then EA ST36; and (7) α-BGT group: α-BGT injection before hemorrhage. Morphological changes in enteric glial cells (EGCs) were observed by immunofluorescence, and glial fibrillary acidic protein (GFAP; a protein marker of enteric glial activation) was evaluated using reverse transcriptase polymerase chain reaction and western blot analysis. Intestinal cytokine levels, gut permeability to 4-kDa fluorescein isothiocyanate (FITC)-dextran, and the expression and distribution of tight junction protein zona occludens (ZO)-1 were also determined. RESULTS: EGCs were distorted following hemorrhage and showed morphological abnormalities. EA ST36 attenuated the morphological changes in EGCs at 6 h, as compared with the VGX, α-BGT and HS groups. EA ST36 increased GFAP expression to a greater degree than in the other groups. EA ST36 decreased intestinal permeability to FITC-dextran (760.5 ± 96.43 ng/mL vs 2466.7 ± 131.60 ng/mL, P < 0.05) and preserved ZO-1 protein expression and localization at 6 h after hemorrhage compared with the HS group. However, abdominal VGX and α-BGT treatment weakened or eliminated the effects of EA ST36. EA ST36 reduced tumor necrosis factor-α levels in intestinal homogenates after blood loss, while vagotomy or intraperitoneal injection of α-BGT before EA ST36 abolished its anti-inflammatory effects. CONCLUSION: EA ST36 attenuates hemorrhage-induced intestinal inflammatory insult, and protects the intestinal barrier integrity, partly via activation of EGCs.

Electroacupuncture ST36 prevents postoperative intra-abdominal adhesions formation.

BACKGROUND: We have recently proved electroacupuncture (EA) ST36 exerted an anti-inflammatory effect in the early phase of intra-abdominal adhesion formation. Evidences indicate that the anti-inflammatory effect of EA ST36 involves a cholinergic anti-inflammatory pathway-dependent mechanism via the vagus nerve. However, the exact effects and accurate vagal modulation of acupuncture in prevention of postoperative intra-abdominal adhesion formation has not been thoroughly evaluated. MATERIALS AND METHODS: Sprague-Dawley rats subjected to abdominal adhesion lesions operation at the cecum and abdominal wall were randomly divided into six groups as follows: (a) EAN: EA non-channel acupoints; (b) EA: EA ST36 after abdominal lesions; (c) VGX/EA: vagotomy (VGX) after abdominal lesions, then EA ST36; (d) VGX/EAN: VGX after abdominal lesions, then EAN; (e) α-BGT/EA: intraperitoneal injection of α-bungarotoxin (α-BGT, an antagonist of α7 subunit of cholinergic nicotinic receptor) before EA ST36, and (f) α-BGT/EAN group: α-BGT injection before EAN. Seven days after abdominal surgical lesions, the levels of tumor necrosis factor-α (TNF-α) and vascular endothelial growth factor (VEGF) in the adhesive tissue were evaluated, macroscopic observation and histopathologic evaluation of adhesion formation and assessment of angiogenesis by immunohistochemical staining of platelet endothelial cell adhesion molecule-1 (CD31) were performed. RESULTS: EA ST36 reduced TNF-α and VEGF levels in adhesive tissue homogenates 7 d after surgery, whereas vagotomy or intraperitoneal injection of α-BGT before EA ST36 reversed its suppressive effects. EA at non-channel acupoints with or without vagotomy or intraperitoneal injection of α-BGT before EA had no suppressive effects on TNF-α and VEGF levels. EA ST36 alleviated the adhesion formation, with both of macroscopic and histopathologic adhesion scores significantly lower than those of the EAN group (1.56 ± 0.29 versus 3.00 ± 0.82, 1.35 ± 0.4 versus 3.91 ± 0.8, respectively, both P < 0.05). Compared with the EAN group, EA ST36 significantly decreased angiogenesis evidenced by reduced CD31 positive microvessel density in adhesive tissue. CONCLUSIONS: EA ST36 might reduce the postoperative local inflammatory response, attenuate the angiogenesis, and alleviate the adhesion formation partly via activating the cholinergic anti-inflammatory mechanism.

Extracellular deposition of mouse senile AApoAII amyloid fibrils induced different unfolded protein responses in the liver, kidney, and heart.

Mouse senile amyloidosis is a disorder in which apolipoprotein A-II deposits extracellularly in many organs as amyloid fibrils (AApoAII). In this study, we intravenously injected 1 µg of isolated AApoAII fibrils into R1.P1-Apoa2(c) mice, to induce AApoAII amyloidosis. We observed that the unfolded protein response was induced by deposition of AApoAII amyloid. We found that the mRNA and the protein expression levels of heat shock protein A5 (HSPA5; also known as glucose-regulated protein 78) were increased in the liver with AApoAII amyloid deposits. Immunohistochemistry showed that HSPA5 was only detected in hepatocytes close to AApoAII amyloid deposits. Furthermore, gene transcription of several endoplasmic reticulum (ER) stress-related proteins increased, including eukaryotic translation initiation factor 2 alpha kinase 3 (Eif2ak3), activating transcription factor 6 (Atf6), activating transcription factor 4 (Atf4), X-box-binding protein 1 splicing (Xbp1s), DNA-damage inducible transcript 3 (Ddit3), and autophagy protein 5 (Atg5). Moreover, apoptosis-positive cells were increased in the liver. Similar results were seen in the kidney but not in the heart. Our study indicates that ER stress responses differed among tissues with extracellular AApoAII amyloid fibril deposition. Although upregulated HSPA5 and the activated unfolded protein response might have roles in protecting tissues against aggregated extracellular AApoAII amyloid deposition, prolonged ER stress induced apoptosis in the liver and the kidney.